The main theme of this proposal remains to study cellular (cell specific) control of virus replication. We propose to examine both host and viral components involved in this control. Our focus will be to examine the host control of viral and cellular DNA replication in linkage to cellular differentiation. 1. Analysis of cell cultures systems able to differentiate and replicate Py. We will expand these studies to include mouse adipocytes and keratinocytes in addition to myoblasts. Both host and viral factors controlling polyomavirus replication during the terminal differentiation will be examined by histochemical and biochemical means. Our focus is the control of DNA replication. We will continue to investigate claims that viral (T-Ag) and host (Rb) genes can compel differentiated myoblasts to reenter the cell cycle. 2. Establish both causality and mechanism of differentiation linked virus replication. We will test the idea that 'repair like' DNA synthesis we have reported (subgenomic, aphidicolin resistant) is involved in terminal differentiation or cell specific Py DNA replication. Molecular genetic approaches will be used to select cells for expression or inhibition of various genes which may affect the ability of myoblasts or adipocytes to terminally differentiate. We will then determine if a corresponding effect is seen on viral and host DNA synthesis. We will also evaluate if recombinant adenoviral vectors can be used both in culture and in vivo to affect differentiation and viral and host DNA synthesis. 3. Expand our in vivo analysis of Py replication in specific mouse tissues. Using various strains of mice we will continue to examine the physiological basis of cell specific in vivo restriction on Py persistence, replication and reactivation and links to cell proliferation and differentiation. We will expand our efforts to include a skin model, whole animal DNA damage and tumor bearing animals and compare in vivo observations to in culture systems. We will also use more sensitive in situ hybridization methods to identify the cellular sites of Py persistence and reactivation. 4. Biochemical analysis of Py and cellular replication occurring during cellular damage and differentiation. We will isolate and characterize the cellular DNA that is made in terminally differentiating cells. We will also use permeabilized cell systems to biochemically characterize the replication enzymes involved in differentiation linked Py and cellular DNA synthesis. Finally we will use 2D gel analysis to determine the mechanism of Py replication in differentiating and damaged cells, both in culture and in permeabilized systems.